The strategies above involve 5 steps that together comprise a push-pull approach. First, we optimize the antigen to improve immunogenicity by epitope enhancement, increasing affinity for MHC. We applied this to 2 new prostate cancer antigens, TARP and POTE. We published a phase I/II TARP clinical trial in D0 prostate cancer patients using a TARP peptide epitope-enhanced to improve HLA-A2 binding and another high-affinity one we mapped. The slope of PSA rise significantly decreased among 72% of 40 patients (p = 0.0012) at 24 wks and 74% (p = 0.0004) at 48 wks, implying slowing of cancer growth. A randomized placebo-controlled phase II trial is now open, with a 2:1 randomization (44:22) of vaccine to placebo. Accrual has been substantially delayed (25 months!) by closure of the NIH Pharmacy Service that necessitated cGMP production and vialing of replacement peptides. The second step is increasing T cell avidity, needed for effective clearance of virus or cancer. We found that lowering antigen dose with a novel adjuvant allowed induction of higher avidity more protective CD4 T cells. The third step is to push the response with molecular adjuvants, such as cytokines, Toll-like receptor (TLR) ligands and NKT agonists, to improve not only the quantity but also the quality of the response. We published that IL-15 is a key mediator of CD4 T cell help for CD8 T cells and that IL-15 increased CD8 T cell avidity. We translated this to humans showing that IL-15 could substitute for CD4 help to induce a primary in vitro CD8 T cell response of naive T cells, and restored responsiveness of CD8 T cells from HIV-infected patients to normal levels. We also found that IL-1beta as adjuvant could enhance CD8 T cell responses and skew CD4 help to Th17. We found surprisingly that the Th17 CD4 cells were not good helpers for CD8 T cell responses as measured by IFN-g production, but rather skewed the CD8 response to IL-17 production through an effect on DCs dependent on IL-21 & 23. We also investigated TLR ligands as adjuvants to mature DCs and induce production of IL-12 and IL-15. We identified in mice a synergistic triple TLR ligand combination and tested this with IL-15 as vaccine adjuvants in a peptide-prime, MVA-boost mucosal vaccine for SIV in macaques, challenging intrarectally with SIVmac251. Only macaques receiving both showed partial protection. In the adaptive immune arm, only polyfunctional CD8 T cells specific for SIV antigens correlated with protection. In the innate immune arm, the adjuvants induced long-lived protection by APOBEC3G. The adjuvants also increased gut CD4 cell preservation, independent of viral load. Adjuvant alone plus a PD-1 blocker and an NKT cell agonist induced CD8-dependent protection (after intrarectal SIV challenge). Yet vaccine could induce MDSC counteracting vaccine efficacy. SIV infection also paradoxically reduced MDSC in bone marrow and increased them in the blood. We also discovered that MDSCs could be infected by SIV. The fourth step is to target the immune response to the relevant tissue, the mucosa in the case of HIV. We published a novel nanoparticle approach for vaccine delivery to the large intestine, using coated vaccine nanoparticles to allow oral delivery and release of the particles primarily in the colon, bypassing the stomach and small intestine and substituting for intrarectal delivery to protect against rectal or vaginal viral challenge. This novel approach allows selective oral delivery to the small or large intestine, enabling selective immunization in these compartments for the first time. We have recently adapted this approach to non-human primates in an AIDS vaccine. 2/7 animals so immunized were protected from acquisition of SHIVsf162P4 high dose rectal challenge (p=0.04 vs 0/29 controls). An expanded study showed 42% vaccine efficacy by an oral nanoparticle vaccine incorporating an Env-CD4D1-D2 fusion protein (FLSC) and MVA against repeated low-dose intrarectal SHIV challenge. Among naive control animals from different sources, colorectal inflammatory target cells and gut microbiomes determined susceptibility to infection. If the small & large intestine are distinct compartments, homing of T cells must be different. We found that homing to the large intestine is governed by DCs from colon, using a mechanism involving alpha4beta7 but not CCR9, distinct from that in the small intestine. In contrast to previous belief, we discovered that CD103+ DCs patrolled the colon lumen in crypts associated with colon patches, attracted by CCL20, to capture and retrieve bacteria. We also published, contrary to accepted dogma, that the type 2 mucosa of the vagina can serve as an inductive site for priming of naive CD8 T cells without help from draining lymph nodes. The fifth step is to remove the brakes, i.e., block negative regulation inhibiting immunity. We previously discovered a new immunoregulatory pathway involving NKT cell suppression of tumor immunity, dependent on IL-13 and TGF-beta. Type I NKT cells (using an invariant TCRa chain) protected, whereas type II NKT cells (using diverse TCRs) suppressed immunity, and these subsets cross-regulated each other, defining a new immunoregulatory axis that could influence subsequent adaptive immune responses. We published that 2 distinct regulatory cells (Tregs & type II NKT) suppressed immunity to the same tumor independently, and found that a third T cell, the type I NKT cell, determines the balance between these, regulating the regulators. As humans with cancer often have a deficiency of type I NKT cell function, they may require blockade of both T regs and type II NKT cells to reveal tumor immunity. We developed a way to make sulfatide-loaded CD1d multimers to stain type II NKT cells, allowing their detection, and found that they are large granular lymphocytes arising independently of PLZF predominantly in the lung and liver, frequent sites of tumor metastases. Unlike type I NKT cells, we found surprisingly that type II NKT cells develop in CD1d-/- mice, but are not functional. Many also express the cKit receptor. Conversely, stimulating with a type I NKT cell agonist can protect against tumors. We discovered a new class of NKT agonist, beta-mannosylceramide, that protects against cancer by a mechanism different from that of the a-GalCer, being dependent on TNF-a and nitric oxide synthase rather than on IFN-g, and that does not induce long-term anergy like a-GalCer. B-ManCer synergizes with a cancer vaccine in mice and stimulates human NKT cells, suggesting translation to human cancer therapy. We are studying it in combination with an intratumoral cancer therapy that induces immunogenic cell death and tumor immunity. A key mediator of the NKT regulatory pathway and an important regulator of T regulatory cells is TGF-b. We found that blockade of TGF-b protected against certain tumors in mice, and synergized with cancer vaccines in 2 mouse models, dependent on CD8 T cells. We translated this into a clinical trial of a human anti-TGF-b monoclonal antibody, and published that in melanoma it is safe and has some anti-cancer activity as a single agent. We found that blocking TGF-b1&2 is sufficient without TGF-b3, and synergizes with anti-PD-1 and a cancer vaccine to treat established murine tumors. Finally, we found that an adenovirus vaccine expressing the extracellular & transmembrane (ECTM) domains of HER-2 can cure large established breast cancers and lung metastases in mice. Protection depends on antibodies inhibiting HER-2 function, and is FcR independent, unlike Herceptin. We made a similar cGMP recombinant adenovirus expressing the human HER-2 ECTM domains used to transduce autologous DCs as a vaccine in a clinical trial in HER-2+ cancer patients, and already at the 2nd & 3rd dose levels, 5/11 evaluable patients with advanced metastatic cancers had clinical benefit.